Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer.

Purpose: Telomerase is a specialized ribonucleoprotein complex that stabilizes telomeres by adding "TAG" repeats to the end of chromosomes. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Telomeres and telomerases play an important role in the longevity of cell and are known to conform "immortalization" on neoplastic cells. Although there exists a lot of information on telomerase in oral cancer, very little is known about their expression in leukoplakia and oral submucous fibrosis (OSF). This study addresses this lacuna. Materials and Methods: In this preliminary study, immunohistochemistry (IHC) was used to detect the expression of hTERT protein in oral squamous cell carcinoma (OSCC) (n=30), leukoplakia (n=15), OSF (n=15) and normal oral mucosa (n=10). The cellular localization of immunostain, intensity of stain, mean nuclear labeling index (LI) and mean nuclear labeling score (LS) of hTERT protein were studied. A total number of 1000 cells were counted in each slide. All the data were analyzed using SPSS software version 10.0.2. The cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity and LI were compared across the groups using Pearson's χ2 test. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant. Results: The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) and from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00). The mean nuclear labeling score was observed to increase from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) (P=0.00) and from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00). Conclusion: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.

Expression of iNOS message in human buccal mucosal [keratinocytes] cell line TR146 using RT-PCR and immunocytochemistry

This study was carried out at the department of Oral Pathology, Queen Mary College of Medicine and Dentistry, Barts and The London to determine whether human oral buccal mucosal [keratinocytes] cell line, TR 146, expressed iNOS message and whether the expression of iNOS is varied when TR146 cells were exposed to different cytokines such as IL-15, IL-8, IL-1 beta, or TNF-alpha using RT-PCR and Immuno-cytochemistry and to determine the effect of the above mentioned cytokines on NO function by measuring nitrite production in TR146 cells. Immuno-cytochemistry analysis revealed that TR146 cells expressed iNOS proteins when incubated with IL-15 and IL-8 and a modest increase was seen with IL-1 beta /TNF-alpha. RT-PCR for iNOS indicated a marked increase in expression when the cells were exposed to IL-8, IL-15 or IL-1beta/ TNF-alpha. NOS activity was assessed by measuring nitrite activity. It was observed that treating the cells with cytokines caused significant increase in nitrite levels, except in the case of IL-8. These results suggest that TR146 cells expressed iNOS, the levels of which varied with various cytokines. It is clear that IL-15, IL-1beta /TNF-a and IL-8 are regulators of iNOS expression in oral keratinocytes and affect NOS activity

Single exposure of human oral mucosa fibroblasts to ultraviolet B radiation reduces proliferation and induces COX-2 expression and activation

The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV) sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX)-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM), primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001). HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05). The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

Matrix metalloproteinase-1 expression in oral submucous fibrosis: An immunohistochemical study.

Context: Oral submucous fibrosis (OSF) is a form of pathological fibrosis affecting the oral mucosa. There is compelling evidence to implicate the habitual chewing of areca nut with the development of OSF. Because collagens are the major structural components of connective tissues, including oral submucosa, the composition of collagen within each tissue needs to be precisely regulated to maintain tissue integrity. Arecoline stimulates fibroblasts to increase the production of collagen by 150%. Aim: As the role of collagenase is implicated in cleaving the collagen under physical conditions, this study was carried out to evaluate the role of collagenase-1 (matrix metalloproteinase [MMP]-1) in a pathologic condition like OSF. Settings and Design: A total of 40 patients were included in the study, comprising of 30 OSF as Group 1 and 10 normal buccal mucosa tissue as Group 2. Materials and Methods: Both the groups were stained for MMP-1 by the immunohistochemical method using the streptavidin HRP-biotin labeling technique. MMP-1 expression intensity in the epithelium and connective tissue was decreased in Group 1 when compared to Group 2. Statistical Analysis Used: Chi-square test of association was used to determine the difference in the expression of MMP-1 between OSF and normal buccal mucosa and among different histological gradings of OSF. Results: The results were statistically significant. However, there was no statistically significant difference between the expression of MMP-1 among different histological grades of OSF in Group 1.

Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis.

OBJECTIVE: We tested the hypothesis that inducible nitric oxide synthase (iNOS) modulates angiogenesis in human models and this information could be extrapolated in elucidating the pathophysiology of oral submucous fibrosis (OSF). A hypothesis which looks inadequate, but is deep rooted in literature is the epithelial alteration ("atrophy") seen in OSF and the events that lead to its causation. This aspect was tried to be addressed and an alternative pathogenetic pathway for the disease is proposed. MATERIALS AND METHODS: This immunohistochemical study sought to investigate the expression of iNOS in OSF samples (n=30) a using monospecific antibody (SC- 2050, Santa Cruz Biotechnology, Inc) to the protein and also to correlate it with different grades of epithelial dysplasia associated with the disease. Twenty (20) healthy adults acted as controls. RESULTS: iNOS staining was not demonstrated in normal oral epithelium. In oral epithelial dysplasia, staining was seen in all cases (100%) in the basal layers of the epithelium and in 30% of cases it extended into the parabasal compartments as well. iNOS staining was uniformly positive in moderate dysplasia with an increase in intensity and distribution noted as the severity of dysplasia progressed. There were highly significant differences in overall positivity for iNOS in epithelium between cases and controls (Mann-Whitney U=11.000, Wilcoxon W=221.00, P=0.000). Significant comparisons were made of mild Vs moderate dysplasia (Mann-Whitney U=48.000, P=0.014) CONCLUSIONS: This study supports our earlier morphological assessment (image analysis) of the nature of vascularity in OSF mucosa. The significant vasodilation noticed in these cases argues against the concept of ischemic atrophy of the epithelium. This observation of vascularity and iNOS expression helped to explain the vasodilation noticed (sinusoids) in this disease; NO being a net vasodilator. The mechanism of activation of iNOS in dysplasia is difficult to explain. The role of contingent paracrine-activating factors on keratinocytes and macrophages is discussed.

Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in oral submucous fibrosis.

Immunohistochemical staining of formalin fixed, paraffin embedded tissue sections of OSF for MMPs-1,2,9 and their tissue inhibitors TIMP-1and 2 was performed using monospecific antibodies coupled with gelatin zymography (MMP-2 and 9) for measuring enzymatic activity quantitatively and for distinguishing the active from the inactive variants of enzymes. The present study, contrary to earlier reports, recorded statistically significant increase in the levels of stromal expression of MMP-1, MMP-2 and MMP-9 and TIMP-1 and TIMP-2 using monospecific antibodies reacting against tissue antigens.The simultaneous increase in reactivity of MMPs and TIMPs poise difficulty in interpretingthe results of this study. The possible reasons for this result, against the backdrop of existing knowledge, were attempted in this study.

Oral epithelial alkaline phosphatase in various phases of menstrual cycle.

We report here on the changes in the Alkaline Phosphatase staining reaction in oral mucosa of women in various phases of menstrual cycle. It appears that the highest reaction for alkaline phosphatase is shown just after ovulation (about 15th-16th day of cycle). It is possible to judge the period of ovulation by taking daily smears and staining them for Alkaline phosphatase.